Calcium Ions Positively Modulate Follicle-Stimulating Hormone- and Exogenous Cyclic 3′,5′-Adenosine Monophosphate-Driven Transcription of the P450scc Gene in Porcine Granulosa Cells1

• Published in: Endocrinology (Philadelphia) Vol. 141; no. 7; pp. 2377 - 2384
• Main Authors: Jayes, F. C. L, Day, R. N, Garmey, J. C, Urban, R. J, Zhang, G, Veldhuis, J. D
• Format: Journal Article
• Language: English
• Published: Endocrine Society 01-07-2000
• DOI: 10.1210/endo.141.7.7558
• EISSN: 1945-7170
• ISSN: 0013-7227

Given the evident modulation of FSH-induced steroidogenesis by
Ca2+ in granulosa cells, we here test the hypothesis that
Ca2+ controls expression of the enzymatically rate-limiting
cytochrome P450scc (CYP11A) gene. To test this postulate,
we quantitated the ability of Ca2+ to regulate: 1)
transcriptional activity of a transiently transfected luciferase
reporter gene driven by a 2.32-kb 5′-upstream fragment of the porcine
P450scc gene promoter region; and 2) accumulation of
endogenous P450scc transcripts in primary monolayer
cultures of porcine granulosa cells. To this end, granulosa cells were
stimulated for 4 h with FSH (15 ng/ml, NIDDK-oFSH-20) or
8-Bromo-cAMP (8 Br-cAMP, 1 mm) in serum-free medium
containing either 1.8 mm Ca2+ or no added
Ca2+ with 100 μm EGTA or 100 μm
CoCl2. In the presence of extracellular Ca2+,
FSH and 8 Br-cAMP stimulated expression of the transfected
P450scc promoter-reporter fusion construct by 5.6 ±
1.1 and 3.6 ± 0.67-fold, respectively over
Ca2+-containing unstimulated control (P≤
0.04, n = 5–6 experiments). The foregoing two agonists
augmented 4-h progesterone production by cultured granulosa cells by
1.8 ± 0.11 and 1.6 ± 0.16-fold, respectively
(P ≤ 0.001 for FSH and P ≤
0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also significantly elevated
endogenous P450scc transcript levels as measured by
homologous solution-hybridization RNase protection assay;
i.e. by 3.1 ± 0.49 and 2.9 ± 0.45-fold,
respectively (P ≤ 0.001). In
Ca2+-free/EGTA-supplemented medium, basal luciferase
reporter-gene activity and endogenous P450scc messenger RNA
accumulation in granulosa cells declined to 34 ± 12% and 78±
12%, respectively, of corresponding values in control (unstimulated
Ca2+-containing) cultures. Extracellular Ca2+
deprivation inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on
P450scc promoter-luciferase reporter expression to 58±
30% (and 58 ± 23%), and restrained endogenous
P450scc message accumulation to 86 ± 15% (and
96 ± 18%) of the value in Ca2+-containing control.
Extracellular Ca2+ withdrawal suppressed FSH (and 8
Br-cAMP)-driven progesterone production over 4 h to basal levels
but did not alter FSH-stimulated cAMP accumulation by granulosa cells.
Ca2+-deprived cells exposed to serum-containing media
regained P450scc responsiveness to both agonists.
Antagonism of cellular uptake of Ca2+ and other divalent
cations via administration of cobalt chloride (100 μm)
inhibited FSH and 8 Br-cAMP’s stimulation of endogenous (but not
exogenous promoter-driven) P450scc gene expression. In
contrast, granulosa-cell concentrations of messenger RNA’s encoding
sterol-carrier protein-2 (SCP-2) and the low density lipoprotein
receptor were not altered by Ca2+ withdrawal.
In summary, uptake of extracellular Ca2+ by porcine
granulosa cells significantly potentiates transactivation of the
endogenously expressed and exogenously transfected P450scc gene by FSH
and 8 Br-cAMP. The agonistic impact of Ca2+ on
P450scc promoter activity is requisite downstream of
FSH-induced cAMP second-messenger signaling.